Skin colonization by S. aureus is common in atopic dermatitis (AD) and is associated with increased disease severity. Both allergic skin inflammation and S. aureus skin colonization influence the immune response to cutaneously introduced antigen. Thus, AD patients are susceptible to cutaneously acquired viral infections, and the incidence of eczema heperticum correlates with disease severity and serum IgE level. Through the ADRN, we have shown that inoculation of vaccinia virus (VV) at the site of allergic skin inflammation results in increased viral spread compared and skews the immune response of mice to VV towards Th2 and Th17. Moreover, AD patients have a reduction in the T cell response to transcutaneous (TC) vaccination, with Yellow Fever vaccine that correlates with IgE levels, and in the antibody response to TC vaccination with Fluzone that correlates with S. aureus skin colonization. We have shown that epicutaneous (EC) sensitization by application of antigen to mouse skin after tape stripping, a surrogate for scratching, results in allergic skin inflammation with many features of AD, and a systemic Th2 response. Our preliminary data shows that co-application of staphylococcal enterotoxin B (SEB) aggravates the allergic skin inflammation and Th2 response to antigen induced by EC sensitization Mechanical skin injury by tape stripping during EC sensitization caused keratinocytes to release the epithelial cytokines TSLP, IL-25 and IL-33, induced rapid expression of IL-4 and IL-13 by innate immune skin cells and resulted in IL-4/IL-13-dependent polarization of skin DCs to promote a Th2- immune response by nave CD4+ cells. We will test the overall hypothesis that that S. aureus skin colonization in the setting of AD-like Th2 allergic skin inflammation results in an altered immune response to cutaneous antigen exposure using TC immunization to influenza virus as a representative model system. We will first test the hypothesis that TC immunization in mice elicits immune responses comparable to, or stronger, than those elicited by IM immunization to influenza. We will then test the hypothesis that S. aureus skin colonization and AD-like allergic skin inflammation, individually and/or in synergy, alter the immune response to influenza TC immunization. We will then investigate the mechanisms by which the immune response to TC vaccination is altered by examining the migration function and polarization of skin DCs, determining the role of epithelial cytokines, and of Th2 cytokines and their sources in the skin, in the Th2 polarization of the immune response to TC vaccination. The proposed studies synergize with the studies in this U19 on TC vaccination of AD patients with a human influenza vaccine (Fluzone), and the clinical trials with blocking antibodies to IL-4R? and TSLP in AD.